Human mycoplasmas cause chronic and opportunistic infections and may also contribute to the pathogenesis of other microbial agents. Indeed, epidemiological studies reveal that M. penetrans is predominantly identified in HIV-infected individuals, which coupled to the observation that M. penetrans-derived lipoproteins stimulate HIV-1 replication, raises the possibility that this Mycoplasma may be a contributory co-pathogen in AIDS progression. Analysis of the complete genome sequence of M. penetrans discloses the presence of 50 lipoprotein genes, 38 of which constitute a large paralogous family (designated mpl) that includes the abundant lipoproteins P35 and P42. Although the precise function of the mpl family is unknown, the individual members are considered to be contingency genes;highly mutable surface protein-encoding sequences with the demonstrated propensity to exhibit high frequency phase variation. Such stochastic changes in lipoprotein expression reflect an adaptive strategy to generate surface diversity within a propagating population of organisms. Phase variation of P42 results from the molecular inversion of a 135-bp DNA cassette that contains the promoter of the lipoprotein gene. Re-orientation of this invertible locus both precludes P42 transcription and creates a putative terminator. Remarkably, analogous cassettes are located proximal to 33 of the mpl genes, suggestive of each contingency gene being subject to phase variation and of the stochastic assortment of combinatorial expression profiles generating a billion potential antigenic variants within the population. Although a number of promoter inversion systems have been characterized in mycoplasmas, only a single invertible promoter is present to mediate transcription of a single gene within a family, following appropriate promoter juxtaposition i.e. a single cell predominantly expresses only one member of a gene family. In contrast, the expansive mpl repertoire with multiple, closely-related invertible segments allowing combinatorial expression, is unprecedented in mycoplasmas and greatly exceeds the largest reported prokaryotic system of 13 segments in Bacteroides fragilis. Furthermore, the invertible promoter is similar in size to the smallest known unit (119 bp) and may be approaching the limit compatible with contraposition. Critical to this proposal, the recombinase that mediates mpl molecular gymnastics has not been identified. Accordingly, to understand the molecular events that govern this strategy of pathoadaptation, it is proposed to identify the recombinase and characterize the interaction with the cognate DNA binding site. The Specific Aims integrate genomic, molecular, biochemical and microbiological approaches to i) identify the recombinase by functional expression in an inversion assay in both E. coli and in a recently developed Mycoplasma host-vector system, ii) delineate the sequence requirements that permit inversion of the promoter region. This proposal addresses a unique phase variation scenario in an enigmatic Mycoplasma and will provide new insight into antigenic diversity generating systems. Data generated by completion of the Specific Aims may provide a platform from which strategies for immune or chemotherapeutic intervention can be devised and implemented. PUBLIC HEALTH RELEVANCE: The proposed research is expected to reveal a critical component of the ability of Mycoplasma penetrans, an AIDS-associated bacterium, to adapt and survive during human infection. The study of the system of antigenic variation in this organism will both further our understanding of this complex process and provide a platform for the future development of strategies for treatment and prevention.